There is extensive evidence demonstrating that angiogenesis, the process of new blood vessel growth, is essential for the growth of solid tumors and their metastases. Blood capillaries are primarily composed of endothelial cells which are usually quiescent under normal physiological conditions. In response to external stimuli, quiescent endothelial cells can degrade the basement membrane via extracellular matrix proteases that permit the endothelial cell to extravasate, and to invade the stromal space and basement membrane. These cells are then capable of changing their morphology, proliferating and forming neovessels (Folkman, J. and Y. Shing. 1992. J. Biol. Chem. 267:10931-10934). To stimulate angiogenesis, tumors induce a variety of factors, including fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Many malignant tumors, however, also generate inhibitors of angiogenesis, including angiostatin and endostatin (O'Reilly, M. S. et al. 1994. Cell 79:315-328; O'Reilly, M. S. et al. 1997. Cell 88:277-285). This complex process implies the presence of multiple controls which can be turned on and off within a short period of time to regulate the angiogenic process.
The process of angiogenesis is tightly regulated by both negative and positive feedback control factors and the result is a homeostatic condition. Imbalance of these factors under pathological conditions can lead to development and progression of disease processes such as tumor growth, diabetic retinopathy, tissue and organ malfunction, and cardiovascular disorders (Folkman, J. 1995. Nat. Med. 1:27-31). Angiostatin is a powerful negative regulator of angiogenesis and potentially can inhibit the growth of primary tumors and metastases in mice (Sim, B. K. et al. 1997. Cancer Res. 57:1329-1334).
Angiostatin was purified from the urine of mice bearing a Lewis lung carcinoma and was identified as a 38 kDa internal fragment of plasminogen (amino acids 98-440) that constituted the first four kringle's of the molecule. Angiostatin can also be generated in vitro by limited proteolysis of plasminogen. Angiostatin produced by various methods has been shown to regress tumor growth in mice (Gately, S. et al. 1997. Proc. Natl. Acad. Sci. USA 94:10868-10872; Gately, S. et al. 1996. Cancer Res. 56:4887-4890; Stathakis, P. et al. 1997. J. Biol. Chem. 272:20641-20645; Dong, Z. et al. 1997. Cell 88:801-810). When tested in vitro, angiostatin has been shown to inhibit cell proliferation, migration, and three-dimensional capillary tube formation in collagen gels, all of which are essential steps in angiogenesis. In vivo, angiostatin is generated by hydrolysis of plasminogen with a number of enzymes including metalloproteinase from macrophages, plasmin reductase, MMP-7 (matrix metalloproteinase 7), gelatinase B/type IV collagenases, MMP-9 (matrix metalloproteinase 9), and pancreatic elastase. Angiostatin has also been shown to efficiently inhibit growth of a broad spectrum of murine and human tumor models in mice (O'Reilly, M. S. et al. 1996. Nat. Med. 2:689-692; O'Reilly, M. S. et al. 1994. Cold Spring Harbor Symp. Quant. Biol. 59:471-482; Wu, Z. et al. 1997. Biochem. Biophys. Res. Commun. 236:651-654; Griscelli, F. et al. 1998. Proc. Natl. Acad. Sci. USA 95:6367-6372) by inhibiting the neovascularization of the tumor, while plasminogen itself failed to inhibit angiogenesis and tumor regression. These data indicate that the specific conformation of the kringle domain is important for angiostatin's anti-angiogenic activity.
Despite its potent anti-cancer value, however, the mechanism of angiostatin's actions and the identity of its cellular receptor are not well defined.
Annexin II is one of the most abundant endothelial cell fibrinolytic receptors for plasminogen and plasminogen activator (Hajjar, K. A. et al. 1994. J. Biol. Chem. 269:21191-21197; Kang, H. M. et al. 1999. Trends Cardiovasc. Med. 9:92-102). Annexin II organizes the assembly of tissue plasminogen activator and plasminogen on its surface. This tri-molecular assembly on endothelial cells can produce plasmin at maximum efficiency in the vascular bed (Hajjar, K. A. et al. 1994. J. Biol. Chem. 269:21191-21197; Felez, J. et al. 1996. Thromb. Haemost. 76:577-584; Kassam, G. et al. 1998. Biochemistry 37:16958-16966). This cell surface-produced plasmin is protected from inactivation. Plasmin is a broad spectrum trypsin-like serine protease that degrades fibrin and several endothelial cell matrix proteins like laminin, thrombospondin, and collagens. The proteolytic degradation of extracellular matrix and dissolution of basement membrane is necessary for endothelial and tumor cells to invade, migrate and promote angiogenesis and metastasis (Kang, H. M. et al. 1999. Trends Cardiovasc. Med. 9:92-102).